Fig 1: DLX3 directly transactivates the expression of Enam, Amelx, and Odam by binding to the enhancer regions.(A) Bioinformatic analysis was performed on 6000 bp 5’-flanking regions of Enam, Amelx, Klk4, and Odam. 1 or 2 conserved DLX3 response elements were separately found on the four genes (translation start site defined as +1 bp). RE, response element. (B) ChIP assays determined whether DLX3 was recruited to the predicted enhancer sites. Left panel: gel images of PCR products, representative of three independent experiments. Right panel: statistics of the PCR results. Values are from three independent experiments, and shown as mean ± SD. *P <0.05 vs. IgG group. (C) Luciferase assays were performed to evaluate the impact of DLX3 on the transcriptional activity of each constructed luciferase reporter, which contained enhancer sequences of specific potential DLX3 response elements. Data are compared with the control group (con), and presented as mean% ± SD. *P <0.05. pEnam-E1 represents pGLEnam-E1, etc. (D) Mutations of DLX3 binding sites reduced the activation effect of DLX3 on the transcriptional activity of the pGLEnam-E1, pGLAmelx-E2, and pGLOdam-E2 reporters. The data represent the mean% ± SD of three independent experiments, each experiment performed in triplicate. *P <0.05 vs. the control (con) group. mut pEnam-E1 represents Mut pGLEnam-E1, etc.
Fig 2: Expression patterns of Dlx3 and EMP genes at the mRNA level during amelogenesis.Real-time RT-PCR was used to analyze the expression levels of Dlx3 and EMP genes using RNA extracted from tooth germs at the indicated stages. The expression level at PN1 was set at 1, and the fold-changes at other stages were calculated relative to PN1. The data represent three independent experiments, and are shown as mean ± SD. *P <0.05 vs. the control (PN1 group). Enam, Enamelin; Amelx, Amelogenin; Ambn, ameloblastin; Tuft, Tuftelin-1; Mmp20, Matrix metalloproteinase 20; Klk4, kallikrein 4; Odam, Odontogenic ameloblast-associated protein; Amtn, Amelotin.
Fig 3: Regulation of Enam, Amelx, Klk4, and Odam by DLX3-overexpression and knockdown.(A) Over-expression of DLX3 was determined by real-time RT-PCR and western blot. Values are mean ± SD of the data from three independent experiments. *P <0.05 vs. control (con) group. (B) After 36 h of transfection, the expression of EMP genes was assessed. The mRNA expression of Enam, Amelx, Klk4, and Odam were significantly up-regulated. Values are presented as mean ± SD. *P <0.05 vs. the control (con) group. (C) After 48 h of transfection, elevated protein expression of ENAM, AMELX, and KLK4 were detected by western blot. Upper panel: western blot bands of ENAM, AMELX, and KLK4 (representative of three independent experiments). Lower panel: densitometric analysis of images of 3 independent experiments. *P <0.05 vs. the control (con) group. (D) Expression levels of DLX3 were determined after transfection with 3 independent Dlx3-specific small interfering RNAs (Dlx3 siRNA #1, #2, #3), or a nonspecific siRNA, NS siRNA. Values are from three independent experiments, and shown as mean ± SD. *P <0.05 vs. NS group. (E) After DLX3 silencing (using Dlx3 siRNA #3), the expression of EMP genes was assessed. The expression of Enam, Amelx, Klk4, and Odam was significantly down-regulated, and then successfully rescued by transfection of plasmid pCI-neo-V5DLX3WT. Values are mean ± SD of data from three independent experiments. *P <0.05. (F) At the protein level, expression of ENAM, AMELX, and KLK4 were also down-regulated by DLX3-knockdown and then rescued by pCI-neo-V5DLX3WT transfection, as analyzed by western blot. Upper panel: western blot bands of ENAM, AMELX, and KLK4 (representative of three independent experiments). Lower panel: densitometric analysis of images of 3 independent experiments. *P <0.05.
Fig 4: Immunostaining of DLX3 protein at different stages of mouse amelogenesis.(A-L) Immunostaining of DLX3 in sections of mouse molar germs at postnatal days 1 (A-C), 3 (D-F), 7 (G-I), and 14 (J-L). Representative figures from three independent experiments are shown. (M) The immunoreactivity of DLX3 protein within ameloblast cells was expressed as the mean optical density of the ameloblasts [integrated optical density (IOD)/unit area]. Each bar represents mean ± SD. *P <0.05 vs. the control (PN1 group). A, D, G, and J, original magnification ×100; B, E, H, and K, high-power magnification of the boxed areas in the low-magnification images, original magnification ×200; C, F, I, and L, high-power magnification of ameloblasts from the area inside the boxes in B, E, H, and K, original magnification ×1000. PA, pre-secretory ameloblasts; SA, secretory ameloblasts; MA, maturation ameloblasts; Od, odontoblast; P, dental pulp; SR, stellate reticulum layer. PN, postnatal day.
Fig 5: TDO mutation of DLX3 inhibits the activation of EMP genes by wild-type DLX3.(A) The plasmids pCI-neo (control empty vector, con), V5DLX3WT, and V5DLX3TDO were transfected into LS8 cells. Equal amounts of V5DLX3WT or V5DLX3TDO plasmid were added to each group, and the total amounts of plasmid in the groups were kept constant using pCI-neo. Over-expression of V5DLX3WT or/and V5DLX3TDO were analyzed by western blot using antibody against V5-tag. A representative figure from three independent experiments is shown. (B) After over-expression, the mRNA expression levels of Enam, Amelx, Klk4, and Odam were evaluated. Values were obtained from three independent experiments, and are presented as mean ± SD. *P <0.05 between the DLX3WT group and the DLX3WT+DLX3TDO group. (C) Left panel: western blot for the protein expression levels of ENAM, AMELX, and KLK4 after transfection (representative of three independent experiments). Right panel: densitometric analysis of images of 3 independent experiments. *P <0.05 between the DLX3WT group and the DLX3WT+DLX3TDO group. (D) After co-transfection with pCI-neo (empty control vector), V5DLX3WT, V5DLX3TDO, or both V5DLX3WT and V5DLX3TDO plasmids, the relative transcriptional activity of the 5 reporter constructs containing potential DLX3 response elements were analyzed by luciferase assays. Data were compared with the control group (con), and are presented as mean% ± SD. *P <0.05 between the DLX3WT group and the DLX3WT+DLX3TDO group. pEnam-E1 represents pGLEnam-E1, etc.
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